1. Field of the Invention
The invention relates to photoluminescent analysis and more particularly to a method and apparatus for determining and correcting for background luminescence in measurements of the amount of luminescent target material.
2. Prior Art
Many substances found in nature are naturally photoluminescent, especially those of organic origin. Thus, quantitative photoluminescent methods are plagued with the problem of ambient photoluminescence interfering with measurements of low concentrations of photoluminescent target substances. It is true that some benefits can be gained by preferentially exciting photoluminescence at the optimum wavelengths for target excitation and observing photoluminescence at the optimum wavelengths for target emission. However, this technique is somewhat limited as the concentration of target molecules is decreased. This is because, for most substances, excitation bandwidths and photoluminescent emission bandwidths are very broad, typically 500 A.degree. and broader, and furthermore there are usually many ambient substances with excitation and emission overlap with target molecules. Because of the superposition of the excitation and emission spectra of many species, the spectrum of the entire ambient sea of substances is usually much broader than 500 A.degree..
This problem has to some extent been solved in U.S. Pat. No. 4,058,732, wherein a target is tagged with a photoluminescent tag with photoluminescent lifetime long compared with ambient photoluminescent substances, and a detection system is gated on only after photoluminescence from ambient substances has decayed. Using that system in combination with rare earth chelates, the range of photoluminescent detectability of rare earths in the presence of background substances has been extended by orders of magnitude.
At this new level of detectability the limiting factor is ambient photoluminescence which is long lived in comparison with ambient fluorescence. At such decay times where the fluorescence of ambient substances has certainly decayed some very weak phosphorescence and "delayed fluorescence" remains. Now, phosphorescence and delayed fluorescence are phenomena which are mostly quenched in liquid systems at room temperature. Nevertheless, at extremely low target concentrations, say 10.sup.-9 or 10.sup.-10 g/cc, ambient luminescent substances which may be of much higher concentrations can give competitive long lived luminescent signals even though they are substantially quenched. See Parker, Photoluminescence in Solutions, Elsevier, 1968, for discussions of residual long lived photoluminescence in solutions. Another source of weak long lived background signals is the phosphorescence of the solid matrix of the sample support or container. These two sources of long lived photoluminescence are the factors which are presently preventing the attainment of further sensitivity.
The benefits of increased sensitivity can be projected to be of importance whether considering the detectability limits on a pure fluorophore or a target tagged with a fluorophore. In the former case, improved sensitivity would be of value in the method of using dyes as tracers to measure the flow of fluids. In that technique a fixed amount of dye is put into the fluid at one point, and the concentration of dye measured downstream at later times. This enables a calculation of the rate that fluid moves in the system. By improving the sensitivity of fluorophore detection, one conserves the amount of dye needed for the measurement. In the case of targets tagged with a fluorophore, clinical tests for substances in bodily fluids are becoming more widely used in medical diagnosis, and the demands for increased sensitivity are following the need to determine substances which are present in smaller quantities. In both of these examples, there are background substances which limit the sensitivity of fluorometric measurement of a fluorescent target.
In dealing with the interference from background substances, it is sometimes possible to determine the background signal in the absence of the fluorophore. Where the test procedure allows this, prior art techniques have been established in which a reading is taken in the appropriate wavelength range of the system minus the fluorophore and then again with the fluorophore, and the first reading is subtracted from the second to obtain the true reading due to the fluorophore. However, there are cases where it is impossible or inconvenient to use this prior art subtraction technique. For example, in the case of fluid flow measurement mentioned above, samples without fluorophores may be long distances away from the point of measurement, and background may be different at these distant points. Or in the clinical test lab, reagents which are added to bodily fluids as part of the test procedure may produce some background photoluminescence in themselves, or may affect the level of other background fluorescence which was present prior to their addition. Thus, a method which could determine the level of background signals in the presence of the fluorophore and test reagents is desirable.
Accordingly, it is an object of the present invention to correct for ambient photoluminescent signals in the wavelength and time domain, and in the presence of fluorescent target substances and associated reagents, in order to extend the limits of fluorescent detectability in the presence of ambient substances beyond their present limits.